This invention relates generally to non-naturally-occurring nutritional compositions for amelioration of disruption of energy metabolism secondary to stress comprising a flavonoid, or derivative thereof, and a synergist in effective amounts. Synergists include amino acids, carbohydrates, carnitines, flavonoids, nucleosides and tocopherols. The invention also relates to non-naturally-occurring compositions comprising an optimized formulation for amelioration of disruption of energy metabolism secondary to stress comprising a flavonoid, or a derivative thereof, and an additional compound in effective amounts. The invention also relates to methods of making such a composition. The invention also relates to methods of ameliorating disruption of energy metabolism secondary to stress, comprising administering to a subject such a composition.
Metabolic pathways are of two types: anabolic, which are involved in synthetic work and require energy; and catabolic, which are degradative and energy-releasing. Catabolic and anabolic pathways can share a common partial sequence, which functions in one direction for synthesis and in the opposite direction for degradation. However, one route is never exactly the reverse of the other, since both need to be exergonic in their respective directions. For example, the pathways for glucose synthesis (gluconeogenesis) and glucose degradation (glycolysis) share many reactions in common, but each have several unique steps. These unique steps generally ensure thermodynamic irreversibility and can serve as regulatory sites. In the catabolic pathways, a substrate is sequentially degraded, releasing energy in the form of ATP (adenosine triphosphate). Catabolic pathways include both the anaerobic pathway (i.e., fermentation) and the aerobic pathway (i.e., oxidative metabolism or respiration). For reviews, see Atkinson (1977) Cellular Energy Metabolism and Its Regulation, Academic Press, New York; Hochachka et al. (1993) Surviving Hypoxia: Mechanisms of Control and Adaptation, CRC Press, Inc., Fl.; and Alberts et al. (1994) Molecular Biology of the Cell, Garland Publ., New York. While metabolic pathways produce ATP, which is an essential energy carrier, by-products of these pathways include free radicals, which are potent cellular injurants.
Reactive oxygen species (ROS), also designated free radicals, include, among other compounds, singlet oxygen, the superoxide anion (O2*xe2x88x92), nitric oxide (NO*), and hydroxyl radicals. Mitochondria are particularly susceptible to damage induced by ROS, as these are generated continuously by the mitochondrial respiratory chain. See, for example, Boveris et al. (1973) Biochem. J. 134:707-716; Turrens et al. (1997) Biosc. Rep. 17:3-8; Tangeras et al. (1980) Biochim. Biophys. Acta 589:162-175; Minotti et al. (1987) Free Radic. Biol. Med. 3:379-387 and Hermes-Lima et al. (1995) Mol. Cell. Biochem. 145:53-60. Free radicals attack membrane lipids and lipoproteins, generating carbon radicals. These in turn react with oxygen to produce a peroxyl radical, which may attack adjacent fatty acids to generate new carbon radicals. This process can lead to a chain reaction producing lipid peroxidation products. Halliwell (1994) Lancet 344:721-724. Damage to the cell membrane can result in loss of cell permeability, increased intercellular ionic concentration, and/or decreased ability to excrete or detoxify waste products. The peroxynitrite anion (ONOOxe2x88x92), a reaction product of O2*xe2x88x92 and nitric oxide (NO*) (Pryor et al. (1995) Am J. Physiol. 268:699-722), appears to be responsible for many effects previously attributed to NO*. Castro et al. (1994) J. Biol. Chem. 269:29409-29415; Ischiropoulos et al. (1992) Arch. Biochem. Biophys. 2:446-453, Halliwell et al. (1995) Ann. Rheumat. Dis. 54:505-510, Salvemini et al. (1996a) Br. J Pharmacol. 118:829-838, Salvemini et al. (1996b) Eur. J. Pharmacol. 303:217-220, Cuzzocrea et al. (1998) Free Radic. Biol. Med. 24:450-459, Wizemann et al. (1994) J. Leukoc. Biol. 56:759-768 and Szabo et al. (1997) J. Clin. Invest. 100:723-735. ROS can also contribute to damage to organs and organisms. These conditions include cell aging, as well as inflammation and cancer.
Free radicals are also problematic in organ transplantation, during which process cells and tissues experience hypoxia. After transplantation, the grafted tissue is reperfused with oxygenated blood. When reperfusion occurs and the flow of oxygen is restored, a burst of free radicals forms. The accumulation of free radicals contributes to post-transplantation injury in tissue giving rise to an increased number of damaged cells and an enhanced immune response by the recipient host. Zhao et al. (1996) J. Neurosci. Res. 45:282-288; Unruh (1995) Chest Surg. Clin. N. Am. 5:91-106. This immune response can lead to inflammation and reduced function in the transplanted tissue and/or rejection and failure of the graft.
Production of ROS also increases when cells experience a variety of stresses, including organ ischemia and reperfusion (as described above) and ultraviolet light exposure and other forms of radiation (Reiter et al. (1998) Ann. N.Y. Acad. Sci. 854:410-424; Saini et al. (1998) Res. Comm. Mol. Pathol. Pharmacol. 101:259-268; Gebicki et al. (1999) Biochem. J. 338:629-636). ROS are also produced in response to cerebral ischemia, including that caused by stroke, traumatic head and spinal injury. In addition, when metabolism increases or a body is subjected to extreme exercise, the endogenous antioxidant systems are overwhelmed, and free radical damage can take place. Free radicals are reported to cause the tissue-damage associated with some toxins and unhealthful conditions, including toxin-induced liver injury. Obata (1997) J. Pharm. Pharmacol. 49:724-730; Brent et al. (1992) J. Toxicol. Clin. Toxicol. 31:173-196; Rizzo et al. (1994) Zentralbl. Veterinarmed 41:81-90; and Lecanu et al. (1998) Neuroreport 9:559-563. Exposure to hyperoxia also results in free-radical production, which can lead to lung damage if not counteracted by sufficient levels of antioxidants. Jenkinson (1989) Clin. Chest Med. 10:37-47. Free radicals may also be responsible for freezing stress in plants. Tao et al. (1998) Cryobiology 37:38-45.
In addition to stresses described above, cells are subject to other stresses, including hyper- and hypothermia, infection, osmotic, hyper- and hypo-gravity, starvation, growth in various reactors (such as bioreactors, fermentation, food preparation, etc.), toxicity (e.g., inhalation of toxic gases such as HCN, phosphates, thiophosphates), drug overdoses, and the like. Common to many of these stresses are injuries secondary to disruptions in energy metabolism. Treatment of these types of injuries includes administration of various individual or combinations of agents which protect against disruptions of energy metabolism and the resulting cell injury during stress (xe2x80x9ccytoprotectantsxe2x80x9d). For example, the time that mammalian cells can undergo stress induced energy dysfunction can be extended by administration of purine derivatives, alone or in combination with electron acceptor compounds and/or amino acids. U.S. Pat. No. 5,801,159.
Because of the potentially damaging nature of free radicals, and because O2*xe2x88x92 generation is continuous, the body has a number of antioxidant defense mechanisms including, but not limited to, enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, NADP transhydrogenase, and thiol peroxidase SP-22, vitamin E, vitamin C, copper and iron transport, storage proteins, water-soluble and lipid-soluble molecular antioxidants, glutathione, NADPH and mitochondrial respiration. Watabe et al. (1997) Eur. J. Biochem. 249:52-60; Guidot et al. (1995) J. Clin. Invest. 96:1131-1136, Radi et al. (1991) J. Biol. Chem. 261:14081-14024. Superoxide radicals produced by the respiratory chain are readily dismutated by mitochondrial superoxide dismutase (MnSOD), leading to the production of H2O2. Fridovich et al. (1997) J. Biol. Chem. 272:18515-18517.
Among the compounds identified with antioxidant activity are some belonging to a group of naturally occurring phenylchromones known as flavonoids. Flavonoids, found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine, are important to the flavor and color of their sources. For a review, see Croft (1998), p. 435-442, in Towards Prolongation of the Healthy Life Span, ed. Harman et al., Annals of the New York Academy of Sciences, New York. Evidence that diets rich in fruits and vegetables appear to protect against cardiovascular disease and some forms of cancer has lead to an interest in the biological effects of flavonoids.
Flavonoids are polyphenolic substances based on a flavan nucleus, comprising 15 carbon atoms, arranged in three rings as C6xe2x80x94C3xe2x80x94C6. Flavonoids are biosynthetically derived from acetate and shikimate such that the A ring has a characteristic hydroxylation pattern at the 5 and 7 position. The B ring is usually 4xe2x80x2, 3xe2x80x24xe2x80x2, or 3xe2x80x24xe2x80x25xe2x80x2-hydroxylated. Flavonoids have generally been classified into 12 different subclasses by the state of oxidation and the substitution pattern at the C2-C3 unit. These subclasses include flavanones (found in citrus fruits), flavones, flavonols (e.g., quercetin; found in onions, olives, tea, wine and apples), anthocyanidins (found in cherries, strawberries, grapes and colored fruits), chalcones, dihydrochalcones, aurones, flavanols, dihydroflavonols, proanthocyanidins (flavan-3,4-diols), isoflavones and neoflavones. Thus far, more than 10,000 flavonoids have been identified from natural sources. Berhow (1998) pp. 67-84 in Flavonoids in the Living System, ed. Manthey et al., Plenum Press, NY.
Flavonoids may act as antioxidants by a number of potential pathways including, but not limited to, free radical scavenging (in which the polyphenol can break the free radical chain reaction) and interactions between flavonoids and phenolic acids with other physiological antioxidants (such as ascorbate or tocopherol). For a compound to be defined as an antioxidant, it must fulfill two conditions: first, when present at low concentrations relative to an oxidizable substrate, it can significantly delay or prevent oxidation of the substrate and second, the resulting radical formed on the polyphenol must be stable so as to prevent it from acting as a chain-propagating radical. Halliwell et al. (1995) Food Chem. Toxicol. 33:601-607. The stabilization is generally through delocalization, intramolecular hydrogen bonding, or by further oxidation by reaction with another lipid radical. Shahidi et al. (1992) Crit. Rev. Food Sci. Nutr. 32:67-103.
In addition to anti-oxidant activity, flavonoids have been reported to possess other biological activities including antihelminthic, antimicrobial, antimalarial, antineoplastic, cytotoxic, mutagenic, carcinogenic, anti-carcinogenic and pro-oxidant action. Recently, compositions containing flavonoids have been described for use in the treatment of damaged or diseased skin and other keratinous tissue. See, for example, U.S. Pat. Nos. 5,945,409 and 5,952,373. Certain redox-active flavonoids appear to be capable of inducing oxidative stress. Thus, relative concentrations of reactants may influence the direction of the anti-oxidant versus pro-oxidant reactions for a flavonoid capable of both donating electrons to electrophilic radicals, terminating radical propagation, and donating electrons to metal ions in the presence of O2, generating ROS. See, for example, Hodnick et al. (1998) pp. 131-150 in Flavonoids in the Living System, ed. Manthey et al., Plenum Press, N.Y.
As mentioned above, ROS contribute to cellular, tissue, organ and organism damage associated with inflammation, hyperthermia, radiation, ischemia, and other unhealthful conditions. However, for many of these conditions, it is not yet clear if ROS are the sole (or even principal) mechanism for inducing damage. Antioxidants, at least in formulations that have been presently tested, are not universally effective cytoprotectants. In some circumstances, for example, antioxidants Vitamin E, probucol and 1,2-Diselenolane-3-pentanoic acid are unable to protect membranes from lipid peroxidation. Kubo et al. (1997) Br. J. Nutr. 78:655-669; Bonnefont-Rousselot et al. (1999) Radiat. Res. 151:343-353; and Matsugo et al. (1997) Biochem. Biophys. Res. Comm. 240:819-824. Antioxidant vitamin E fails to prevent hyperoxic lung injury in premature animals. Langley et al. (1 992) Comp. Biochem. Physiol. Comp. Physiol. 103:793-799. Most antioxidants also had no protective effect against free radical production by rose bengal photoactivation in perfused hearts. Bernier et al. (1991) Free Radic. Biol. Med. 10:287-296.
Common to many of the stresses described herein are injuries secondary to disruptions in energy metabolism. The need remains for identification of effective, novel formulations and combinations of compounds, particularly but not limited to combinations that may be used as medical foods or dietary supplements, which aid in the survival and recovery of cells during injury secondary to stress and disruption of energy metabolism.
In one embodiment, the invention relates to non-naturally-occurring compositions or formulations, particularly nutritional formulations, for amelioration of disruption of energy metabolism secondary to stress comprising a flavonoid and a synergist. The synergist is selected from the group consisting of amino acids, carbohydrates, carnitines, flavonoids and nucleosides, and the flavonoid and the synergist are present in amounts effective to ameliorate the disruption of energy metabolism. When a second flavonoid is the synergist, the first flavonoid and the second flavonoid are different. The primary flavonoid may also be a combination of different flavonoids.
In another embodiment, the invention relates to non-naturally-occurring compositions comprising an optimized formulation for amelioration of disruption of energy metabolism secondary to stress comprising a first compound comprising a flavonoid, or derivative thereof, and a second compound. The second compound is selected from the group consisting of amino acids, carbohydrates, carnitines, flavonoids and nucleosides and derivatives thereof, and the flavonoid and the second compound are present in amounts effective to ameliorate said disruption of energy metabolism secondary to stress. When a second flavonoid is the second compound, the first flavonoid and the second flavonoid are different.
In another embodiment, the invention relates to a method for amelioration of disruption of energy metabolism secondary to stress, comprising administering to a subject a non-naturally-occurring composition comprising a flavonoid and a synergist selected from the group consisting of amino acids, carbohydrates, carnitines, flavonoids and nucleosides.
In another embodiment, the invention encompasses a method of making a non-naturally-occurring composition to effect amelioration of disruption of energy metabolism secondary to stress comprising adding a flavonoid and a synergist selected from the group consisting of amino acids, carbohydrates, carnitines, flavonoids and nucleosides.
In various embodiments, the flavonoid is the flavanone hesperetin. The flavonoid can be in the form of a pharmaceutically acceptable salt.
In various embodiments, the synergist can be in the form of a pharmaceutically acceptable salt.
In various embodiments, the synergist is an amino acid selected from the group consisting of glycine, alanine, and N-acetyl-cysteine.
In various embodiments, the synergist is a carbohydrate selected from the group consisting of fructose-1,6-bisphosphate, galactose, ADP-ribose, hydroxybutyrate, pyruvate and ribulose.
In various embodiments, the synergist is a carnitine selected from the group consisting of carnitine tartrate, acetyl carnitine and carnitine free base.
In various embodiments, the synergist is a nucleoside selected from the group consisting of adenosine and inosine.
In various embodiments, the synergist is a flavonoid selected from the group consisting of chrysin, diosmin, hesperidin, luteolin, rutin, and quercetin.
In additional embodiments, the synergist is a tocopherol, such as alpha-, delta-or gamma tocopherol.
In various embodiments, the stress can be induced by an environmental alteration, chemical insult or physiological condition. Environmental alterations include, but are not limited, to hypothermia, hyperthermia, hypoxia, and ionizing radiation. Chemical insults include, but are not limited to, drug toxicity, chemotherapy, exposure to at least one toxin, and cell culture. Physiological conditions include, but are not limited to, physical exertion, aging, disease and pre-surgical and post-surgical situations.
Non-naturally-occurring optimized compositions for amelioration of disruption of energy metabolism are also contemplated by the present invention. Such compositions are combinations of compounds in amounts determined or predicted to be particularly effective to ameliorate the disruption of energy metabolism secondary to stress. In optimized formulations, the combined amounts of flavonoid and the additional compound are selected to increase, augment or enhance the cytoprotective effect of either agent when used individually at the same concentration.